The present invention is generally directed to cancer diagnosis or prognosis, and more particularly to the detection of mutation(s) in blood cell-free DNA using primer extension and PCR.
The presence of abnormally high levels of cell-free blood DNA (CFDNA) in the plasma/serum of cancer patients was demonstrated in 1977. However, it is only recently that CFDNA in cancer has attracted attention and that its possible use as a marker for diagnosis or prognosis has been investigated. Mutations in CFDNA have been characterized in a large variety of cancer types and sites, including, for example, colorectal, pancreas, lung, bladder, head and neck and liver cancers. Various types of DNA alterations have been reported in CFDNA, including point mutations, DNA hypermethylations, microsatellite instabilities and losses of heterozygosity. In many instances, these alterations were identical to the ones found in the primary tumor tissue of the patient, supporting the tumoral origin of altered CFDNA.
Occurrence of alterations in CFDNA, as well as increase in the overall level of CFDNA, is not restricted to any particular tumor site, type or grade. However, there is tendency for significantly larger amounts of CFDNA in patients with late stage disease and metastasis. Thus, CFDNA may provide a very valuable source of genetic material as a surrogate for molecular analysis in cancer and pre-cancer patients. CFDNA could be an alternate to tissue biopsy and provide an easily accessible and non-invasive modality to determine the genetic background of materials.
The detection of CFDNA is not without challenges. Tumor DNA is present at very low concentrations in the blood. In addition, the mutation detection should be carried out in the presence of a very large excess of normal homologous DNA. The excess of homologous sequences interfaces with mutation detection by sequencing when mutated DNA is less than 5 percent because the signal from mutated nucleotide will be completely masked by homologous nucleotides from normal DNA. Since the majority of mutation detection techniques are based on sequencing, such methods are not adequate for blood DNA samples. In addition, whole genome sequencing is not applicable either for DNA mutation detection because it requires the sequencing of hundreds of normal genomes to find the mutated genome.
In addition, the modern approach for treatment of cancer known as “personalized medicine” relies on identification of the genetic traits, normal and abnormal, which determines the particular behavior of the same disease in different patients. Based on this information an individualized management can be devised to ensure maximum of benefit with minimum risk for patients.
An integral component of personalized medicine is the targeted therapy aimed at overcoming the effect of specific molecular alterations in an individual tumor.
To ensure the benefit of targeted therapy, testing for mutations in several genes has become a basic requirement. This testing is usually performed on tissue specimen obtained by biopsy at initial diagnosis. However, frequently the amount of biopsy specimen is not sufficient for these studies or diagnosis is made by thin needle aspirates or by cytology, which make the genetic studies very difficult or impossible.
Another difficulty in genetic studies is the frequent contamination of tumoral specimen with normal cells. In such cases, either the patient accepts the inconvenience of a second biopsy or he/she will not benefit from the targeted therapy.
About 40-years ago, it was shown that tumoral, as well as normal cells release their DNA in blood in high molecular weight form and that this nucleic acid caries the same molecular alterations as the cells of origin.
As noted above, the blood cell-free DNA (BCF DNA or CFDNA) has become an attractive source of genetic information, provided an adequate technology is available to bring such data to the clinician. However, the identification of such alterations in blood is difficult because the tumoral DNA is found in very low concentration and it is mixed with a large amount of normal homologous DNA.
When compared to the biopsy source, besides being more accessible and convenient to patients, the plasma DNA could also allow identification of genotype changing during progression through emergency of new mutations in cancer cells. These mutations could herald, for instance, the development of drug resistance, and thus, guide timely changes of treatment, or could identify new potential targets for additional therapy.
Some recent preliminary studies indicate that mutations in BCF could be detected very early during the progression of disease when the cancer can be cured with surgical resection.
This is a new and exciting application of BCF DNA with very important implications for cancer patients. At present, only rarely the cytotoxic or targeted therapy can cure a malignancy. In general, the surgical resection and sometimes radiation therapy can cure at least 50% of patients with any solid tumor, provided the disease is localized in the organ of origin. The lower limit of clinical detection of a malignant lesion is usually a nodule of 1 cm diameter. Such nodule contains about 109 cells from most histological type. It is conceivable that nodule smaller than 1 cm and below the limit of clinical detection, and containing a few hundreds of millions of cancer cells, could still release enough mutated DNA which, when detected, would be indicative of the presence of a developing malignancy. Following the detection of such mutation in BCF DNA, the patient could be followed by PET scans every 4-6 months until the nodule is identified. If this nodule is confirmed malignant by biopsy, the patient can have the nodule resected and eventually be cured of cancer.
With this idea in mind I have developed a novel strategy for mutation detection in BCF DNA that is able to detect very low level of mutated genes.